BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases

Prime editing allows precise installation of any single base substitution and small insertions and deletions without requiring homologous recombination or double-strand DNA breaks in eukaryotic cells. However, the applications in bacteria are hindered and the underlying mechanisms that impede efficient prime editing remain enigmatic. Here, we report the determination of vital cellular factors that affect prime editing in bacteria. Genetic screening of 129 Escherichia coli transposon mutants identified sbcB, a 3ʹ→5ʹ DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli, combinational deletions of which with two additional 3ʹ→5ʹ DNA exonucleases, xseA and exoX, drastically enhanced the prime editing efficiency by up to 100-fold. Efficient prime editing in wild-type E. coli can be achieved by simultaneously inhibiting the DNA exonucleases via CRISPRi. Our results pave the way for versatile applications of prime editing for bacterial genome engineering.


Reviewers' Comments:
Reviewer #1: Remarks to the Author: In this manuscript, the authors explored the applications of prime editing in bacteria.Specifically, through screening 129 Keio collection of E. coli mutants, they identified that 3'-5' DNA exonucleases sbcB, xseA, and exoX are the key factors that inhibit prime editing in several bacterial species.Consistently, deleting these genes or repressing their expression through CRISPRi enabled prime editing in several bacterial strains.Based on this, a 3'-directed hydrolysis model for inhibiting prime editing in bacteria was proposed.This work is original and impressive.I have a few concerns.
Major points: 1.There have been various CRISPR-Cas9, Cas12, and native type I Cascade-mediated genome editing established in prokaryotes.How necessary is it to develop prime editing in bacteria?This point was not well justified in the manuscript.
2. Related to point 1, are there bacterial species/ strains and/or the types of editing in which existing editing strategies and approaches are incapable of and prime editing could achieve?The authors should show some examples of such applications.Alternatively, as a methodology manuscript, applications of the developed technique in representative Gram-positive and Gramnegative bacterial species should be shown.
3. The authors proposed a 3'-directed hydrolysis model for inhibiting prime editing in bacteria just based on the fact that deletion of 3'-5' exonuclease genes improved prime editing.It would better to provide more direct, biochemical evidence for this mechanism.
Minor points: 1. Authors should provide schematic diagrams for the types of editing mentioned in the manuscript, e.g. in the Figure 1, schematic diagrams for the types of editing as depicted in the Xaxises should be provided.2. Overall, the figure legends are too simple.Some necessary details should be provided in the legends so that readers can understand the figures with necessary details without going through the materials and methods section.Reviewer #2: Remarks to the Author: In this work, Zhang and colleagues show that repressing or deleting exonucleases involved in DNA repair can radically boost prime editing in bacteria.After finding that prime editing is highly efficient in M. smegmatis, they began screening for DNA repair factors potentially accounting for poor editing in E. coli.This screen uncovered sbcB, a 3'-to-5' DNA exonuclease, whose deletion boosted prime editing in E. coli across target sites.Further deleting two other known DNA endonucleases (xseA, exoX) further boosted prime editing.Engineering the pegRNA based on prior work further enhanced prime editing, in some cases exceeding 80%.Adopting CRISPRi to repress these three genes allowed enhanced editing in a WT strain of E. coli, while deleting some of these genes in Klebsiella and Acinetobacter also enhanced prime editing across sites.Finally, the authors present a model of how these exonucleases interfere with prime editing.
To my knowledge, prime editing in bacteria has only been reported in one publication (Tong et al., Nat Commun 2021), but with poor editing efficiencies at genomic sites, hindering its adoption by the bacterial community.This work makes an important advance by identifying and removing the responsible barriers, which could open up broad use of prime editing in bacteria and open a range of editing options for fundamental research and strain engineering.The authors further provide extensive datasets, and the manuscript was well-written and should be easily accessible to a broad audience.I do have some important comments about the toxicity/escape as well as claims in the abstract and reproducibility, although I see these only strengthening the work.
Major comments: 1.While the authors provide extensive editing data, the impact of CFUs compared to non-targeting conditions would be incredibly important to report.Targeting, particularly in the absence of repair proteins, could be highly cytotoxic, with few surviving cells.Such a condition would also select for escape phenotypes such as disruption of the Cas9 or the pegRNA or an unintended edit at the target site.While I don't expect the authors to provide this information for all target sites, reporting relative CFUs and looking at whether non-edited cells are escapers for a few representative examples would be incredibly important to potential users.
2. While the main text generally contains conclusions that nicely match the presented data, the abstract takes a number of liberties that need to be rephrased.Specifically, -"Here, we report….":inhibition was only shown for E. coli, so this should indicate inhibition or deletion.
-"Comparative prime editing….":The comparison was only between M. smegmatis and E. coli, so better to state these two species.
-"Genetic screening of….":It would be more pertinent to state the number of repair genes that were screened rather than the number of transposons tested.If the number is smaller, then it's reasonable to drop the specific number.
-"We propose a 3'-directed….":I would recommend rewording this to indicate that disrupting related exonucleases enhanced prime editing rather than claiming the model holds.Otherwise, there is a much higher bar to claim a mechanistic model spanning different bacterial strains.
-"Efficient prime editing can be achieved….":CRISPRi was only shown in E. coli, so it remains to be shown that this same approach can greatly boost prime editing in other bacteria.
3. I could not find any description of the number of replicates or the nature of the replicates.In most cases, the error bars were extremely small, raising some concerns how independent the replicates were and how reproducible the overall results are.
4. The authors note that the ddCas12a silencing construct represses the target genes by ~80% individually.How much is repression when the genes are combined?Individual values normally decrease as multiplexing increases.
Other comments: 5. L. 73-77: the original demonstration of prime editing in E. coli was able to achieve chromosomal editing, albeit at very low levels.Therefore, some rewording is needed here to better capture what was previously demonstrated.
6. L. 118: For comparisons of the WT and ΔmutS (Ext.Data Fig. 6), the authors write that mutS deletions have minimal or no impact on the editing efficiency, but at least for the adhE target site it looks like it could have an effect.Including statistical tests with the corresponding p-values would be helpful.7. L. 122: how many genes were screened as part of the transposon set?It would also be helpful for these genes to be listed in the SI. 8. L. 128-130: The transition from the prior sentence is abrupt.I recommend first stating that sbcB emerged as the top hit in the screens.9. L. 134: For comparisons of the contribution of single-or combined knockouts of the 3′→5′-DNA exonucleases on the prime editing efficiency (e.g., Fig. 2c-g), are the differences significant from the WT values?Here the authors could include statistical tests and add the p-values to the main text.10.L. 139: Which genes were deleted?Listing these here or in the SI would be helpful.11.L. 147: Looking at Fig. S9, the edit made to xylB yields increased editing when sbcB and other repair genes were deleted.Why is there an exception, and can the authors reword the sentence to capture this? 12. L. 210: Replace "significant" with "substantial" since no statistical comparisons are being made.
13. Fig 2a: Write genes following standard nomenclature for bacteria.Also change "scrap" to "scrape".14.Fig. 2b: based on the depicted assay, the axes should indicate the colony ratio rather than the mutation occurrence, as the authors did not directly assess the frequency of this edit.
15. Fig. 2c-g: what are the colored bars meant to represent?16.Fig. 2h: for the middle arrow, can an intermediate step be added to show gap filling?17.I recommend making the plasmids available on Addgene upon publication of the work to ensure the approach can be broadly disseminated.Providing annotated plasmid maps for key constructs such as through Benchling would also ease adoption.
18.The amplicon sequencing data should be made publicly available.
19.The authors could discuss in more detail the importance of investigating redundancy of gene functions in repair pathways as important finding.The authors could also provide a short statement on how prime editing in the field of microbiology could be of advantage in comparison to already established CRISPR-Cas gene editing technologies.
Reviewer #3: Remarks to the Author: This manuscript written by Zhang H et al describes the development of a practical prime editing method, which is useful for bacterial fine genome editing.The authors wanted to provide a useful prime editing method for E. coli and found that Exonuclease I (the sbcC gene product) is critical for reducing the efficiency in E. coli cells.Furthermore, additional mutations of xseA and exoX drastically enhanced the prime editing efficiency (up to 100-fold).From these experimental results, the authors proposed a model of the 3'-directed hydrolysis for degradation of the prime editing intermediates to explain inhibition of prime editing in Bacteria.I think this work is interesting and is probably useful for the researchers studying bacterial genetics to follow their protocol.I have several comments to be addressed before publication.
1. My first impression is that all data are graphs representing genome editing efficiency and none of the raw data is shown.I think actual experimental data before calculations of the editing efficiency should be shown as supplemental data.In addition, I want to see the colonies appearing as Ref resistance on the agar plate from sbcC mutant as compared with that from the wild type.Please see the picture of a representative agar plate.
2. The authors selected 129 mutants of the repair gene from the Keio mutant library, and found that the sbcC mutant was critical for increasing prime editing efficiency.I think the mutant strains selected in this study should be listed up in the manuscript, at least in the supplemental data.3. Their conclusion insists that ExoI (the sbcC product) is the only critical nuclease for the prime editing and ExoVII (the xseA product) and ExoX (the exoX product) can assist the critical function of ExoI in its absence.Deletion of xseA or exoX by theirselves does not affect the prime editing efficiency.From these results, the authors proposed the 3'-directed hydrolysis model.I think additional experiments are needed to make this model credible.The 3' -flapped DNA seems to be much more preferable substrate for ExoI as compared with ExoVII and ExoX.This substrate specificity can be confirmed by in vitro assays.Otherwise, do they have any other idea to explain the difference of three 3'-5' exonucleases?4. A result shown in Fig. 2g is different from others.Deletion of one more gene from xseA and exoX, in addition to sbcC deletion is not different from deletions of all three genes.I understand the locus dependence of the prime editing.However, the result of Fig. 2g shows a completely different characteristics from other loci, and this result affects on the conclusion of this study.The authors should adequately address this issue.
5. Regarding to the result shown in Fig. 4a, the difference of the efficiency by BacPE varies from1% to 89.4%.I think the results are too variable, and therefore, the practicality and versatility of BacPE will be questionable, if it is true.
4. The authors note that the ddCas12a silencing construct represses the target genes by ~80% individually.How much is repression when the genes are combined?Individual values normally decrease as multiplexing increases.

Response:
We assessed the repression efficiency by RT-qPCR in the revised manuscript (Supplementary Figure 10).In the BacPE system, multiple crRNA expression units were assembled into a single plasmid and the repression efficiency of sbcB, xseA and exoX was 57.4%, 88.5% and 76.3%, respectively.A slight decrease in repression efficiency was observed for xseA when multiple crRNAs were expressed simultaneously, whereas no significant decrease in repression efficiency for sbcB and exoX when multiple crRNAs were expressed, likely because the crRNAs were transcribed using separated promoters, rather than transcribed in a crRNA array driven by a single promoter.Other comments: 5. L. 73-77: the original demonstration of prime editing in E. coli was able to achieve chromosomal editing, albeit at very low levels.Therefore, some rewording is needed here to better capture what was previously demonstrated.

Response
Response: We have revised the sentence as "applying prime editors in prokaryotes are limited to Escherichia coli and the editing activities are at low levels" in the revised manuscript (Line 88).
6. L. 118: For comparisons of the WT and ΔmutS (Ext.Data Fig. 6), the authors write that mutS deletions have minimal or no impact on the editing efficiency, but at least for the adhE target site it looks like it could have an effect.Including statistical tests with the corresponding p-values would be helpful.

Response:
The p-values were noted in the revised manuscript.We revised "However, deletion of mutS had minimal or no impact on the editing efficiencies" as "In most cases (40/45), deletion of mutS had minimal or no impact on the improvement of prime editing efficiency" in the revised manuscript (Line 148).
3. What does MMR represent?It should be spelled the first time it appears in the text.4. Legend of Fig 2 c-g, " in at different targeting loci"

Fig. 3
CRISPRi-mediated repression of gene expression with different spacers.Gene-specific spacers were designed to target scbB, xseA or exoX.In the BacPE system, different crRNAs were assembled into a single plasmid to inhibit sbcB, xseA and exoX simultaneously.Student's t-test was performed.Data represent mean ± s.d. of n = 3 independent replicates.